Restriction

modification system

The restriction modification system (RM

system) is found in bacteria and other
prokaryotic organisms, and provides a
defense against foreign DNA, such as that
borne by bacteriophages.

Bacteria have restriction enzymes, also

called restriction endonucleases, which
cleave double stranded DNA at specific
points into fragments, which are then
degraded further by other endonucleases.
This prevents infection by effectively
destroying the foreign DNA introduced by
an infectious agent (such as a
bacteriophage). Approximately one-
quarter of known bacteria possess RM
systems and of those about one-half have
more than one type of system.

As the sequences recognized by the

restriction enzymes are very short, the
bacterium itself will almost certainly
contain some within its genome. In order
to prevent destruction of its own DNA by
the restriction enzymes, methyl groups are
added. These modifications must not
interfere with the DNA base-pairing, and
therefore, usually only a few specific bases
are modified on each strand.

Endonucleases cleave internal/non-

terminal phosphodiester bonds. They do
so only after recognising specific
sequences in DNA which are usually 4-6
base pairs long, and often palindromic.

History
The RM system was first discovered by
Salvatore Luria and Mary Human in 1952
and 1953.[1][2] They found that
bacteriophage growing within an infected
bacterium could be modified, so that upon
their release and re-infection of a related
bacterium the bacteriophage’s growth is
restricted (inhibited) (also described by
Luria in his autobiography on pages 45
and 99 in 1984).[3] In 1953, Jean Weigle
and Giuseppe Bertani reported similar
examples of host-controlled modification
using different bacteriophage system.[4]
Later work by Daisy Roulland-Dussoix and
Werner Arber in 1962[5] and many other
subsequent workers led to the
understanding that restriction was due to
attack and breakdown of the modified
bacteriophage’s DNA by specific enzymes
of the recipient bacteria. Further work by
Hamilton O. Smith isolated HinDII, the first
of the class of enzymes now known as
restriction enzymes, while Daniel Nathans
showed that it can be used for restriction
mapping.[6] When these enzymes were
isolated in the laboratory they could be
used for controlled manipulation of DNA,
thus providing the foundation for the
development of genetic engineering.
Werner Arber, Daniel Nathans, and
Hamilton Smith were awarded the Nobel
Prize in Physiology or Medicine in 1978 for
their work on restriction-modification.

Types
There are four categories of restriction
modification systems: type I, type II, type
III and type IV, all with restriction enzyme
activity and a methylase activity (except
for type IV that has no methylase activity).
They were named in the order of discovery,
although the type II system is the most
common.

Type I systems are the most complex,

consisting of three polypeptides: R
(restriction), M (modification), and S
(specificity). The resulting complex can
both cleave and methylate DNA. Both
reactions require ATP, and cleavage often
occurs a considerable distance from the
recognition site. The S subunit determines
the specificity of both restriction and
methylation. Cleavage occurs at variable
distances from the recognition sequence,
so discrete bands are not easily visualized
by gel electrophoresis.

Type II systems are the simplest and the

most prevalent. Instead of working as a
complex, the methyltransferase and
endonuclease are encoded as two
separate proteins and act independently
(there is no specificity protein). Both
proteins recognize the same recognition
site, and therefore compete for activity.
The methyltransferase acts as a monomer,
methylating the duplex one strand at a
time. The endonuclease acts as a
homodimer, which facilitates the cleavage
of both strands. Cleavage occurs at a
defined position close to or within the
recognition sequence, thus producing
discrete fragments during gel
electrophoresis. For this reason, Type II
systems are used in labs for DNA analysis
and gene cloning.

Type III systems have R (res) and M (mod)

proteins that form a complex of
modification and cleavage. The M protein,
however, can methylate on its own.
Methylation also only occurs on one
strand of the DNA unlike most other
known mechanisms. The heterodimer
formed by the R and M proteins competes
with itself by modifying and restricting the
same reaction. This results in incomplete
digestion.[7][8]

Type IV systems are not true RM systems

because they only contain a restriction
enzyme and not a methylase. Unlike the
other types, type IV restriction enzymes
recognize and cut only modified DNA.[9]

Function
Neisseria meningitidis has multiple type II
restriction endonuclease systems that are
employed in natural genetic
transformation. Natural genetic
transformation is a process by which a
recipient bacterial cell can take up DNA
from a neighboring donor bacterial cell
and integrate this DNA into its genome by
recombination. Although early work on
restriction modification systems focused
on the benefit to bacteria of protecting
themselves against invading
bacteriophage DNA or other foreign DNA,
it is now known that these systems can
also be used to restrict DNA introduced by
natural transformation from other
members of the same, or related species.
In the pathogenic bacterium Neisseria
meningitidis (meningococci), competence
for transformation is a highly evolved and
complex process where multiple proteins
at the bacterial surface, in the membranes
and in the cytoplasm interact with the
incoming transforming DNA. Restriction-
modification systems are abundant in the
genus Neisseria. N. meningitidis has
multiple type II restriction endonuclease
systems.[10] The restriction modification
systems in N. meningitidis vary in
specificity between different clades.[10][11]
This specificity provides an efficient barrier
against DNA exchange between clades.[10]
Luria, on page 99 of his autobiography,[3]
referred to such a restriction behavior as
“an extreme instance of unfriendliness.”
Restriction-modification appears to be a
major driver of sexual isolation and
speciation in the meningococci.[12]
Caugant and Maiden[13] suggested that
restriction-modification systems in
meningococci may act to allow genetic
exchange among very close relatives while
reducing (but not completely preventing)
genetic exchange among meningococci
belonging to different clonal complexes
and related species.

RM systems can also act as selfish

genetic elements, forcing their
maintenance on the cell through
postsegregational cell killing.[14]

Some viruses have evolved ways of

subverting the restriction modification
system, usually by modifying their own
DNA, by adding methyl or glycosyl groups
to it, thus blocking the restriction enzymes.
Other viruses, such as bacteriophages T3
and T7, encode proteins that inhibit the
restriction enzymes.

To counteract these viruses, some

bacteria have evolved restriction systems
which only recognize and cleave modified
DNA, but do not act upon the host's
unmodified DNA. Some prokaryotes have
developed multiple types of restriction
modification systems.

R-M systems are more abundant in

promiscuous species, wherein they
establish preferential paths of genetic
exchange within and between lineages
with cognate R-M systems.[15] Because the
repertoire and/or specificity of R-M
systems in bacterial lineages vary quickly,
the preferential fluxes of genetic transfer
within species are expected to constantly
change, producing time-dependent
networks of gene transfer.
Applications

Molecular biology …

(a) Cloning: RM systems can be cloned

into plasmids and selected because of the
resistance provided by the methylation
enzyme. Once the plasmid begins to
replicate, the methylation enzyme will be
produced and methylate the plasmid DNA,
protecting it from a specific restriction
enzyme.

(b) Restriction Fragment Length

Polymorphisms: Restriction enzymes are
also used to analyse the composition of
DNA in regard to presence or absence of
mutations that affect the specificity of the
REase cleavage specificity. When wild-type
and mutants are analysed by digestion
with different REases, the gel-
electrophoretic products vary in length,
largely because mutant genes will not be
cleaved in a similar pattern as wild-type for
presence of mutations that render the
REases nonb-specific to the mutant
sequence.

Gene therapy …

The bacteria R-M system has been

proposed as a model for devising human
anti-viral gene or genomic vaccines and
therapies since the RM system serves an
innate defense-role in bacteria by
restricting tropism of bacteriophages.[16]
Research is on REases and ZFN that can
cleave the DNA of various human viruses,
including HSV-2, high-risk HPVs and HIV-1,
with the ultimate goal of inducing target
mutagenesis and aberrations of human-
infecting viruses.[17][18][19] The human
genome already contains remnants of
retroviral genomes that have been
inactivated and harnessed for self-gain.
Indeed, the mechanisms for silencing
active L1 genomic retroelements by the
three prime repair exonuclease 1 (TREX1)
and excision repair cross complementing
1(ERCC) appear to mimic the action of
RM-systems in bacteria, and the non-
homologous end-joining (NHEJ) that
follows the use of ZFN without a repair
template.[20][21]

A major advance is the creation of artificial

restriction enzymes created by linking the
FokI DNA cleavage domain with an array
of DNA binding proteins or zinc finger
arrays, denoted now as zinc finger
nucleases (ZFN).[22] ZFNs are a powerful
tool for host genome editing due to their
enhanced sequence specificity. ZFN work
in pairs, their dimerization being mediated
in-situ through the FoKI domain. Each zinc
finger array (ZFA) is capable of recognizing
9-12 base-pairs, making for 18-24 for the
pair. A 5-7 bp spacer between the cleavage
sites further enhances the specificity of
ZFN, making them a safe and more
precise tool that can be applied in
humans. A recent Phase I clinical trial of
ZFN for the targeted abolition of the CCR5
co-receptor for HIV-1 has been
undertaken.[23]

Their relation with mobile

genetic elements (MGEs)
R-M systems are major players in the co-
evolutionary interaction between mobile
genetic elements (MGEs) and their
hosts.[24] Genes encoding R-M systems
have been reported to move between
prokaryotic genomes within MGEs such as
plasmids, prophages, insertion
sequences/transposons, integrative
conjugative elements (ICEs) and
integrons. However, it was recently found
that there are relatively few R-M systems
in plasmids, some in prophages, and
practically none in phages. On the other
hand, all these MGEs encode a large
number of solitary R-M genes, notably
MTases.[24] In light of this, it is likely that R-
M mobility may be less dependent on
MGEs and more dependent, for example,
on the existence of small genomic
integration hotspots. It is also possible
that R-M systems frequently exploit other
mechanisms such as natural
transformation, vesicles, nanotubes, gene
transfer agents or generalized
transduction in order to move between
genomes.

See also
Methylation
Restriction enzyme

References
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